Β-oxidation Pathway Research Articles

Optimizing hexanoic acid biosynthesis in Saccharomyces cerevisiae for the de novo production of olivetolic acid

Medium chain fatty acids (MCFAs) are valuable platform compounds for the production of biotechnologically relevant chemicals such as biofuels and biochemicals. Two distinct pathways have been implemented in the yeast Saccharomyces cerevisiae for the biosynthetic production of MCFAs: (i) the mutant fatty acid biosynthesis (FAB) pathway in which the fatty acid synthase (FAS) complex is mutated and (ii) a heterologous multispecies-derived reverse β-oxidation (rBOX) pathway. Hexanoic acid has become of great interest as its acyl-CoA ester, hexanoyl-CoA, is required for the biosynthesis of olivetolic acid (OA), a cannabinoid precursor. Due to insufficient endogenous synthesis of hexanoyl-CoA, recombinant microbial systems to date require exogenous supplementation of cultures with hexanoate along with the overexpression of an acyl-CoA ligase to allow cannabinoid biosynthesis. Here, we engineer a recombinant S. cerevisiae strain which was metabolically optimized for the production of hexanoic acid via the FAB and rBOX pathways and we combine both pathways in a single strain to achieve titers of up to 120 mg L−1. Moreover, we demonstrate the biosynthesis of up to 15 mg L−1 OA from glucose using hexanoyl-CoA derived from the rBOX pathway.

Sustainable biosynthesis of caproate from waste activated sludge via electro-fermentation: Perspectives of product spectrum, economic and environmental impacts

Electro-fermentation (EF) has emerged as a promising method to produce value-added medium chain fatty acids (MCFAs) via chain elongation (CE). The biorefinery of waste activated sludge (WAS) to MCFAs has been attracting increasing attention. However, so far anaerobic_CE process was commonly employed, while the contribution and mechanism of EF_CE still remain unclear. In the present study, a comprehensive analysis of caproate biosynthesis from prefermented WAS via EF_CE was performed. The reduction in substrates resulted in an increase in caproate production, yielding the maximum caproate (299.8 mg COD/g volatile suspended solid) in the minimum substrate concentration (25 % prefermented WAS, EF13 group). The highest utilization rate (78.76 %) of soluble proteins was also achieved in EF13. Significant positive correlation among caproate yield, electrochemically active bacteria, caproate-synthesizing consortium and homo-acetogen was revealed by molecular ecological network and Mantel test. Further analysis of the metabolic pathways revealed that EF13 demonstrated more key enzymes participated in the production of acetyl-CoA via the Wood-Ljungdahl pathway and the conversation of acetyl-CoA to caproate via the reverse β-oxidation pathway. Moreover, compared to the anaerobic_CE process, the economic benefits of the EF_CE process significantly increased, and the environmental impacts were greatly reduced. The life cycle assessment and economic benefits analysis identified the strengths of EF_CE and proposed a sustainable strategy to facilitate the commercialization of electro-fermentation assisted biorefinery technology.

Combinatorial metabolic engineering strategy of precursor pools for the yield improvement of spinosad in Saccharopolyspora spinosa

Spinosad is an insecticide produced by Saccharopolyspora spinosa, and its larvicidal activity is considered a promising approach to combat crop pests. The aim of this study was to enhance the synthesis of spinosad through increasing the supply of acyl-CoAs precursor by the following steps. (i) Engineering the β-oxidation pathway by overexpressing key genes within the pathway to promote the synthesis of spinosad. The results showed that the overexpression of fadD, fadE, and fadA1 genes, as well as the co-expression of fadA1 and fadE genes, increased the yield of spinosad by 0.36-fold, 0.89-fold, 0.75-fold and 1.25-fold respectively. (ii) Employing combinatorial engineering of the β-oxidation pathway and ACC/PCC pathway to promote the synthesis of spinosad. The results showed that the co-expression of fadE and pccA, as well as accC and fadE, resulted in a 1.77-fold and 1.43-fold increase in spinosad production respectively. (iii) When exogenous triacylglycerol was added to the fermentation medium, the solely engineering of the β-oxidation pathway increased the yield of spinosad by 7.13-fold, reaching 427.23 mg/L. While the combinatorial engineering of both the β-oxidation pathway and ACC/PCC pathway increased the yield of spinosad by 9.61-fold, reaching 625.17 mg/L, and further optimization of the culture medium resulted in an even higher yield of spinosad, reaching 1293.43 mg/L. The results of this study indicate that the above combination strategy can promote the efficient biosynthesis of spinosad.

Combined metabolome and transcriptome analysis reveal the mechanism of water stress in Ophiocordyceps sinensis

BackgroundOphiocordyceps sinensis (O. sinensis) is the dominant bacterium in the asexual stage of Chinese cordyceps, and its growth usually suffers from water stress. Thus, simulating its ecological growth environment is crucial for artificial cultivation. This study aimed to reveal the mechanism underlying the water stress tolerance of Ophiocordyceps sinensis (O. sinensis) by combining metabolomic and transcriptome analyses to identify crucial pathways related to differentially expressed genes (DEGs) and metabolites (DEMs) involved in the response to water stress.ResultsGene coexpression analysis revealed that many genes related to ‘betalain biosynthesis’, ‘tyrosine metabolism’, ‘linoleic acid metabolism’, ‘fructose and mannose metabolism’, and ‘starch and sucrose metabolism’ were highly upregulated after 20d-water stress. Metabolomic analysis revealed that many metabolites regulated by these genes in these metabolic pathways were markedly decreased. On the one hand, we surmised that carbohydrate metabolism and the β-oxidation pathway worked cooperatively to generate enough acyl-CoA and then entered the TCA cycle to provide energy when exposed to water stress. On the other hand, the betalain biosynthesis and tyrosine metabolism pathway might play crucial roles in response to water stress in O. sinensis by enhancing cell osmotic potential and producing osmoregulatory substances (betaine) and antioxidant pigments (eumelanin).ConclusionsOverall, our findings provide important information for further exploration of the mechanism underlying the water stress tolerance of O. sinensis for the industrialization of artificial cultivation of Chinese cordyceps.

Single- vs. two-stage fermentation of an organic fraction of municipal solid waste for an enhanced medium chain carboxylic acids production − The impact of different pH and temperature

Single-stage fermentation was characterized by low medium chain carboxylic acids concentrations and different mesophilic temperatures had little effect on the process performance, whereas thermophilic conditions and pH 5.5 led to lactate and ethanol accumulation. Two-stage fermentation enabled almost twofold increase in the caproate productivity, that reached 0.8 g/L-d (1.7 ± 0.1 gCOD/L-d) with a caproate concentration of 4.0 g/L (8.4 gCOD/L) at specificity of 37% and led to a reduced release of carbon dioxide. Under pH 6.5 and temperature of 37°C the production of medium chain carboxylic acids required higher concentrations of ethanol. Number of genes annotated to fatty acid biosynthesis pathway exceeded the number of sequences assigned to the reverse β-oxidation pathway. Core microorganisms encoding these pathways belonged to Caproiciproducens, Caproicibacterium and Clostridium. This study provides a valuable insight for valorisation of organic fraction of municipal solid waste into carboxylates in a single- and two-stage systems.

A-066 Does essential fatty acid deficiency alter ethanolamine plasmalogen concentrations in red blood cells? Implications for clinical testing

Abstract Background Plasmalogens are glycerophospholipids characterized by a fatty alcohol at the sn-1 position, a polyunsaturated fatty acid at the sn-2 position, most commonly docosahexaenoic acid (DHA) or arachidonic acid, and a polar head group at the sn-3 position, mainly phosphoethanolamine. Markedly reduced plasmalogens are seen in peroxisome biogenesis defects, including Zellweger spectrum disorders (ZSD), since the first two steps of plasmalogen synthesis occur in peroxisomes. Disruption of the peroxisomal β-oxidation pathway also causes DHA deficiency in ZSD patients. Since plasmalogens are composed of fatty acids, the deficiency of DHA or other essential fatty acids could further decrease the plasmalogen level in ZSD patients and lower the results of plasmalogen testing in patients without ZSD. To test this hypothesis, we evaluated the impact of essential fatty acid (EFA) deficiency on plasmalogens in patients with ZSD and individuals with EFA deficiency due to unrelated causes. Methods Red blood cell (RBC) samples were obtained from individuals without ZSD referred to ARUP laboratories for clinical EFA testing (n = 80; age range: birth - 89 years), and from patients with ZSD (n = 7; age range birth - 9 years). Three ZSD patients were older and with a less severe phenotype. A total of 18 intact ethanolamine plasmalogen species were extracted from packed RBCs and quantified using a XEVO TQ-XS Mass Spectrometer in conjunction with Ultra-High Performance Liquid Chromatography (Waters). Total plasmalogen values were also calculated; plasmalogen deficiency was defined as ≤60% of age-matched control median. Fatty acids (FAs) were quantified by gas chromatography negative chemical ionization-mass spectrometry (GC-NCI-MS). EFA deficiency was diagnosed by increased Triene/Tetraene ratio (Mead acid/Arachidonic acid). Data was analyzed using Prism v.8.3.0 software (La Jolla, CA). The study was approved by the Institutional Review Board of the University of Utah. Results Out of the 80 samples from individuals without ZSD referred for EFA testing, 18% had normal fatty acid profile, 57% were receiving dietary supplements and 25% had elevated Triene/Tetraene ratio suggesting EFA deficiency. EFA deficiency was accompanied by low DHA only in two individuals without ZSD, whereas it was present in most of the samples from ZSD patients evaluated (5/7). None of the individuals without ZSD were deficient in plasmalogens. Moreover, when compared to individuals without ZSD with a normal FA profile, there was no significant difference observed with EFA deficiency or FA supplementation. Although our ZSD cohort is small, no correlation between EFA deficiency and plasmalogen levels was also seen in ZSD: 2 patients with milder phenotype had EFA deficiency but normal plasmalogens, 2 patients had EFA deficiency and low plasmalogens, 2 patients with low plasmalogens had low DHA without EFA deficiency, and 1 patient had low plasmalogens but normal EFA. Conclusions Plasmalogen quantitation in RBCs by LC-MS/MS is not significantly affected by EFA deficiency. Although more data is warranted to elucidate the correlation between plasmalogen levels and ZSD phenotype, plasmalogens can be within normal reference intervals in ZSD regardless of EFA status.

Improving Lipid Content in the Diatom Phaeodactylum tricornutum by the Knockdown of the Enoyl-CoA Hydratase Using CRISPR Interference.

The diatom Phaeodactylum tricornutum shows potential as a source for biofuel production because of its considerable lipid content. Fatty acid β-oxidation plays a critical role in lipid breakdown. However, we still have a limited understanding of the role of fatty acid β-oxidation in lipid content in this microalga. In our study, we utilized a CRISPR interference method to reduce the expression of enoyl-CoA hydratase (PtECH), which is involved in the hydration of trans-2-enoyl-CoA to produce 3-hydroxyacyl-CoA during the β-oxidation pathway. Using this method, we developed two transgenic lines, PtECH21 and PtECH1487, which resulted from interference at two different sites of the PtECH gene, respectively. RT-qPCR analysis confirmed that the mRNA levels of PtECH in both mutants were significantly lower compared to the wild type. Surprisingly, the lipid content of both mutants increased notably. Additionally, both knockdown mutants exhibited higher chlorophyll content and improved photosynthetic efficiency of the photosystem II compared to the wild type. This study introduces a new approach for enhancing lipid content in P. tricornutum and expands our knowledge of the functions of enoyl-CoA hydratase in microalgae.

Candidatus Thiothrix phosphatis SCUT-1: A novel polyphosphate-accumulating organism abundant in the enhanced biological phosphorus removal system

A novel coccus Thiothrix-related polyphosphate-accumulating organism (PAO) was enriched in an acetate-fed enhanced biological phosphorus removal (EBPR) system. High EBPR performance was achieved for an extended period (>100 days). A high-quality draft genome (completeness 97.2 %, contamination 3.26 %) was retrieved, representing a novel Thiothrix species (with similarity<93.2 % to known Thiothrix species), and was denoted as 'Candidatus Thiothrix phosphatis SCUT-1'. Its acetate uptake rate (6.20 mmol C/g VSS/h) surpassed most Ca. Accumulibacter and known glycogen-accumulating organisms (GAOs), conferring their predominance in the acetate-fed system. Metatranscriptomic analysis suggested that Ca. Thiothrix phosphatis SCUT-1 employed both low- and high-affinity pathways for acetate activation, and both the conventional (PhaABC) pathway and the fatty acid β-oxidation pathway for PHA synthesis; additionally, a much more efficient FAD-dependent malate: quinone oxidoreductase (MQO) were encoded and employed than the traditional malate dehydrogenase (MDH) to oxidize malate to oxaloacetate in the TCA and glyoxylate cycle, collectively contributing to a higher acetate utilization and processing rate of this microorganism. Batch tests further demonstrated the versatile ability of this PAO in using VFA (acetate, propionate, and butyrate), lactate, amino acids (aspartate and glutamate), and glucose as carbon sources for EBPR, showing a partially overlapped but unique ecological niche of this microorganism comparing to Ca. Accumulibacter and known GAOs. A metabolic model was built for Ca. Thiothrix phosphatis SCUT-1 using the above-mentioned carbon sources for EBPR. Overall, this study represents the first comprehensive characterization of the physiology and metabolic characteristics of representative coccus Thiothrix-related PAOs, which are expected to provide new insights into PAO microbiology in EBPR systems.

Outstanding enhancement of caproate production with microwave pyrolyzed highly reductive biochar addition

The integration of biochar into microbial Chain Elongation (CE) proves to be an effective tool of producing high-value bio-based products. This study innovatively applied biochar fabricated under microwave irradiation with carbon fiber cloth assistance into CE system. Results highlighted that microwave biochar achieved maximal CE efficiency yielding 8 g COD/L, with 3-fold increase to the blank group devoid of any biochar. Microwave biochar also obtained the highest substrate utilization rate of 94 %, while conventional biochar group recorded 90 % and the blank group was of 74 %. Mechanistic insights revealed that the reductive surface properties facilitated CE performance, which is relevant to fostering dominant genera of Parabacteroides, Bacteroides, and Macellibacteroides. By metagenomics, microwave biochar up-regulated functional genes and enzymes involved in CE process including ethanol oxidation, the reverse β-oxidation pathway, and the fatty acid biosynthesis pathway. This study effectively facilitated caproate production by utilizing a new microwave biochar preparation strategy.

Pharmacological targeting of AMPK to restore glucose and fatty acid metabolism homeostasis attenuates transplanted kidney fibrosis

Chronic fibrosis often occurs in transplanted kidneys, leading to progressive functional decline. The underlying mechanisms may involve disruption in the metabolism of renal tubular epithelial cells. The liver kinase B1 (LKB1)-AMPK pathway is a pivotal regulatory hub for glucose and fatty acid metabolism and may play a role in transplanted kidney fibrosis, but it has not been reported. In this study we administered fenofibrate, 2-deoxyglucose, or metformin to modulate metabolism in Brown Norway rat kidney transplants and investigated pathways involved in fibrosis using various assays. We identified an impaired LKB1-AMPK pathway within epithelial cells, resulting in perturbed glucose and fatty acid metabolism, collagen secretion, extracellular matrix remodeling, and epithelial-mesenchymal transition. ACOX1, a pivotal enzyme in the fatty acid peroxisomal β-oxidation pathway, played an important role in transplanted renal fibrosis. Furthermore, several metabolism-targeting drugs, particularly metformin, emerged as potent fibrosis inhibitors. Metformin attenuated fibrosis, improved renal function, and reduced inflammation and macrophage infiltration in the transplanted kidneys. These results provide new perspectives for understanding the complex molecular basis underlying transplanted renal fibrosis and developing novel therapeutic strategies.

Metabolic engineering of Escherichia coli for high-level production of benzyl acetate from glucose

BackgroundBenzyl acetate is an aromatic ester with a jasmine scent. It was discovered in plants and has broad applications in food, cosmetic, and pharmaceutical industries. Its current production predominantly relies on chemical synthesis. In this study, Escherichia coli was engineered to produce benzyl acetate.ResultsTwo biosynthetic routes based on the CoA-dependent β-oxidation pathway were constructed in E. coli for benzyl acetate production. In route I, benzoic acid pathway was extended to produce benzyl alcohol by combining carboxylic acid reductase and endogenous dehydrogenases and/or aldo-keto reductases in E. coli. Benzyl alcohol was then condensed with acetyl-CoA by the alcohol acetyltransferase ATF1 from yeast to form benzyl acetate. In route II, a plant CoA-dependent β-oxidation pathway via benzoyl-CoA was assessed for benzyl alcohol and benzyl acetate production in E. coli. The overexpression of the phosphotransacetylase from Clostridium kluyveri (CkPta) further improved benzyl acetate production in E. coli. Two-phase extractive fermentation in situ was adopted and optimized for benzyl acetate production in a shake flask. The most optimal strain produced 3.0 ± 0.2 g/L benzyl acetate in 48 h by shake-flask fermentation.ConclusionsWe were able to establish the whole pathway for benzyl acetate based on the CoA-dependent β-oxidation in single strain for the first time. The highest titer for benzyl acetate produced from glucose by E. coli is reported. Moreover, cinnamyl acetate production as an unwanted by-product was very low. Results provided novel information regarding the engineering benzyl acetate production in microorganisms.Graphical abstract

Exploring Fatty Acid β-Oxidation Pathways in Bacteria: From General Mechanisms to DSF Signaling and Pathogenicity in Xanthomonas.

Fatty acids (FAs) participate in extensive physiological activities such as energy metabolism, transcriptional control, and cell signaling. In bacteria, FAs are degraded and utilized through various metabolic pathways, including β-oxidation. Over the past ten years, significant progress has been made in studying FA oxidation in bacteria, particularly in E. coli, where the processes and roles of FA β-oxidation have been comprehensively elucidated. Here, we provide an update on the new research achievements in FAs β-oxidation in bacteria. Using Xanthomonas as an example, we introduce the oxidation process and regulation mechanism of the DSF-family quorum sensing signal. Based on current findings, we propose the specific enzymes required for β-oxidation of several specific FAs. Finally, we discuss the future outlook on scientific issues that remain to be addressed. This paper supplies theoretical guidance for further study of the FA β-oxidation pathway with particular emphasis on its connection to the pathogenicity mechanisms of bacteria.

Pseudomonas rhizophila S211 as a microbial cell factory for direct bioconversion of waste cooking oil into medium-chain-length polyhydroxyalkanoates.

The present study examines the use of waste cooking oil (WCO) as a substrate for medium-chain-length polyhydroxyalkanoates (mcl-PHA) production by Pseudomonas rhizophila S211. The genome analysis revealed that the S211 strain has a mcl-PHA cluster (phaC1ZC2DFI) encoding two class II PHA synthases (PhaC1 and PhaC2) separated by a PHA depolymerase (PhaZ), a transcriptional activator (PhaD) and two phasin-like proteins (PhaFI). Genomic annotation also identified a gene encoding family I.3 lipase that was able to hydrolyze plant oils and generate fatty acids as favorable carbon sources for cell growth and PHA synthesis via β-oxidation pathway. Using a three-variable Doehlert experimental design, the optimum conditions for mcl-PHA accumulation were achieved in 10% of WCO-based medium with an inoculum size of 10% and an incubation period of 48h at 30°C. The experimental yield of PHA from WCO was 1.8g/L close to the predicted yield of 1.68 ± 0.14g/L. Moreover, 1H nuclear magnetic resonance spectroscopy analysis confirmed the extracted mcl-PHA. Overall, this study describes P. rhizophila as a cell factory for biosynthesis of biodegradable plastics and proposes green and efficient approach to cooking oil waste management by decreasing the cost of mcl-PHA production, which can help reduce the dependence on petroleum-based plastics.

Whole-cell biocatalysis for phthalate esters biodegradation in wastewater by a saline soil bacteria SSB-consortium

Phthalic acid esters (PAE) are widely used as plasticizers and have been classified as ubiquitous environmental contaminants of primary concern. PAE have accumulated intensively in surface water, groundwater, and wastewaters; thus, PAE degradation is essential. In the present study, the ability of a saline soil bacteria (SSB)-consortium to degrade synthetic wastewater-phthalates with alkyl chains of different lengths, such as diethyl phthalate (DEP), di-n-butyl phthalate (DBP), benzyl butyl phthalate (BBP), and di (2-ethylhexyl) phthalate (DEHP) was characterized. A central composite design-response surface methodology was applied to optimize the degradation of each phthalate, where the independent variables were temperature (21–41 °C), pH (5.3–8.6) and PAE concentration (79.5–920.4 mg L−1), and Gas Chromatography-Mass Spectrometry was used to identify the metabolites generated during phthalate degradation. Optimal conditions were 31 °C, pH 7.0, and an initial PAE concentration of 500 mg L−1, where the SSB-consortium removed 84.9%, 98.47%, 99.09% and 98.25% of initial DEP, DBP, BBP, and DEHP, respectively, in 168h. A first-order kinetic model explained - the biodegradation progression, while the half-life of PAE degradation ranged from 12.8 to 29.8 h. Genera distribution of the SSB-consortium was determined by bacterial meta-taxonomic analysis. Serratia, Methylobacillus, Acrhomobacter, and Pseudomonas were the predominant genera; however, the type of phthalate directly affected their distribution. Scanning electron microscopy analysis showed that high concentrations (1000 mg L−1) of phthalates induced morphological alterations in the bacterial SSB-consortium. The metabolite profiling showed that DEP, DBP, BBP, and DEHP could be fully metabolized through the de-esterification and β-oxidation pathways. Therefore, the SSB-consortium can be considered a potential candidate for bioremediation of complex phthalate-contaminated water resources.

Imatinib therapy of chronic myeloid leukemia significantly reduces carnitine cell intake, resulting in adverse events

ObjectiveA prominent, safe and efficient therapy for patients with chronic myeloid leukemia (CML) is inhibiting oncogenic protein BCR::ABL1 in a targeted manner with imatinib, a tyrosine kinase inhibitor. A substantial part of patients treated with imatinib report skeletomuscular adverse events affecting their quality of life. OCTN2 membrane transporter is involved in imatinib transportation into the cells. At the same time, the crucial physiological role of OCTN2 is cellular uptake of carnitine which is an essential co-factor for the mitochondrial β-oxidation pathway. This work investigates the impact of imatinib treatment on carnitine intake and energy metabolism of muscle cells. MethodsHTB-153 (human rhabdomyosarcoma) cell line and KCL-22 (CML cell line) were used to study the impact of imatinib treatment on intracellular levels of carnitine and vice versa. The energy metabolism changes in cells treated by imatinib were quantified and compared to changes in cells exposed to highly specific OCTN2 inhibitor vinorelbine. Mouse models were used to test whether in vitro observations are also achieved in vivo in thigh muscle tissue. The analytes of interest were quantified using a Prominence HPLC system coupled with a tandem mass spectrometer. ResultsThis work showed that through the carnitine-specific transporter OCTN2, imatinib and carnitine intake competed unequally and intracellular carnitine concentrations were significantly reduced. In contrast, carnitine preincubation did not influence imatinib cell intake or interfere with leukemia cell targeting. Blocking the intracellular supply of carnitine with imatinib significantly reduced the production of most Krebs cycle metabolites and ATP. However, subsequent carnitine supplementation rescued mitochondrial energy production. Due to specific inhibition of OCTN2 activity, the influx of carnitine was blocked and mitochondrial energy metabolism was impaired in muscle cells in vitro and in thigh muscle tissue in a mouse model. ConclusionsThis preclinical experimental study revealed detrimental effect of imatinib on carnitine-mediated energy metabolism of muscle cells providing a possible molecular background of the frequently occurred side effects during imatinib therapy such as fatigue, muscle pain and cramps.

Variability in n-caprylate and n-caproate producing microbiomes in reactors with in-line product extraction.

Medium-chain carboxylates (MCCs) are used in various industrial applications. These chemicals are typically extracted from palm oil, which is deemed not sustainable. Recent research has focused on microbial chain elongation using reactors to produce MCCs, such as n-caproate (C6) and n-caprylate (C8), from organic substrates such as wastes. Even though the production of n-caproate is relatively well-characterized, bacteria and metabolic pathways that are responsible for n-caprylate production are not. Here, three 5 L reactors with continuous membrane-based liquid-liquid extraction (i.e., pertraction) were fed ethanol and acetate and operated for an operating period of 234 days with different operating conditions. Metagenomic and metaproteomic analyses were employed. n-Caprylate production rates and reactor microbiomes differed between reactors even when operated similarly due to differences in H2 and O2 between the reactors. The complete reverse β-oxidation (RBOX) pathway was present and expressed by several bacterial species in the Clostridia class. Several Oscillibacter spp., including Oscillibacter valericigenes, were positively correlated with n-caprylate production rates, while Clostridium kluyveri was positively correlated with n-caproate production. Pseudoclavibacter caeni, which is a strictly aerobic bacterium, was abundant across all the operating periods, regardless of n-caprylate production rates. This study provides insight into microbiota that are associated with n-caprylate production in open-culture reactors and provides ideas for further work.IMPORTANCEMicrobial chain elongation pathways in open-culture biotechnology systems can be utilized to convert organic waste and industrial side streams into valuable industrial chemicals. Here, we investigated the microbiota and metabolic pathways that produce medium-chain carboxylates (MCCs), including n-caproate (C6) and n-caprylate (C8), in reactors with in-line product extraction. Although the reactors in this study were operated similarly, different microbial communities dominated and were responsible for chain elongation. We found that different microbiota were responsible for n-caproate or n-caprylate production, and this can inform engineers on how to operate the systems better. We also observed which changes in operating conditions steered the production toward and away from n-caprylate, but more work is necessary to ascertain a mechanistic understanding that could be predictive. This study provides pertinent research questions for future work.

Long-chain fatty acids facilitate acidogenic fermentation of food waste: Attention to the microbial response and the change of core metabolic pathway under saturated and unsaturated fatty acids loading

Long-chain fatty acids (LCFAs) are recognized as a significant inhibitory factor in anaerobic digestion of food waste (FW), yet they are inevitably present in FW due to lipid hydrolysis. Given their distinct synthesis mechanism from traditional anaerobic digestion, little is known about the effect of LCFAs on FW acidogenic fermentation. This study reveals that total volatile fatty acids (VFAs) production increased by 9.98 % and 4.03 % under stearic acid and oleic acid loading, respectively. Acetic acid production increased by 20.66 % under stearic acid loading compared to the control group (CK). However, the LCFA stress restricted the degradation of solid organic matter, particularly under oleic acid stress. Analysis of microbial community structure and quorum sensing (QS) indicates that LCFA stress enhanced the relative abundance of Lactobacillus and Klebsiella. In QS system, the relative abundance of luxS declined from 0.157 % to 0.116 % and 0.125 % under oleic acid and stearic acid stress, respectively. LCFA stress limited the Autoinducer-2 (AI-2) biosynthesis, suggesting that microorganisms cannot use QS to resist the LCFA stress. Metagenomic sequencing showed that LCFA stress promoted acetic acid production via the conversion of pyruvate and acetyl-CoA to acetate. Direct conversion of pyruvate to acetic acid increased by 47.23 % compared to the CK group, accounting for the enhanced acetic acid production under stearic acid loading. The abundance of β-oxidation pathway under stearic acid loading was lower than under oleic acid loading. Overall, the stimulating direct conversion of pyruvate plays a pivotal role in enhancing acetic acid biosynthesis under stearic acid loading, providing insights into the effect of LCFA on mechanism of FW acidogenic fermentation.

Nano zero valent iron stimulated acetaldehyde oxidation, electron transfer, and RBO pathway for enhanced medium-chain carboxylic acids production

Nano zero-valent iron (NZVI) has been shown to effectively enhance the chain elongation (CE) process, addressing the issue of limited yield of medium-chain carboxylic acids (MCCA) from organic wastewater. However, the specific impact of NZVI on the metabolism of CE bacteria (CEB) is not well understood. In this study, it was aimed to investigate the mechanism by which an optimal concentration of NZVI influences CE metabolism, particularly in relation to ethanol oxidation, electron transfer, and MCCA synthesis. This was achieved through single-factor influence experiments and metagenomic analysis. The results showed that the addition of 1 g/gVSS NZVI achieved the highest MCCA yield (n-caproic acid + n-octanoic acid) at 2.02 g COD/L, which was 4.9 times higher than the control. This improvement in MCCA production induced by NZVI was attributed to several factors. Firstly, NZVI facilitated the oxidation of acetaldehyde, leading to its reduced accumulation in the system (from 18.4 % to 5.8 %), due to the optimized chemical environment created by NZVI corrosion, including near-neutral pH and a more reductive oxidation–reduction potential (ORP). Additionally, the inherent conductivity property of NZVI and the additional Fe ions released during corrosion improved the electron transfer efficiency between CEB. Lastly, both the composition of microbial communities and the abundance of unique enzyme genes confirmed the selective stimulation of NZVI on the reverse β-oxidation (RBO) pathway. These findings provide valuable insights into the role of NZVI in CEB metabolism and its potential application for enhancing MCCA production in CE bioreactors.

2,3,5,4′-tetrahydroxy-stilbene-2-O-β-D-glucoside promotes liver regeneration after partial hepatectomy in mice: The potential involvement of PPARα-mediated fatty acid metabolism

Ethnopharmacological relevance2,3,5,4′-tetrahydroxy-stilbene-2-O-β-D-glucoside (TSG) is the principal bioactive compound contained in Polygonum multiflorum Thunb. (PMT), which is traditionally recorded to possess tonic and anti-aging efficacy. Aim of the studyTo identify the TSG-provided promotion on liver regeneration (LR) following partial hepatectomy (PHx) in mice and to explicate its involved mechanism. Materials and methodsThe promotion of TSG on LR was evaluated by hematoxylin and eosin (H&E), 5-bromodeoxyuridinc (BrdU) and Ki-67 staining, and measuring the level of proliferating cell nuclear antigen (PCNA) and Cyclin D1 in mice with PHx at different time points. Gene Expression Omnibus (GEO, GSE15239) database and the label-free quantitative proteomics from liver of mice at 24 h after PHx were integrated to identify potential involved critical proteins, which were verified by Western-blot, Real-time polymerase chain reaction (RT-PCR), molecular docking and luciferase activity assay. Primary hepatocytes isolated from mice were used to investigate the TSG-provided promotion on proliferation in vitro. ResultsTSG (20 mg/kg) promoted LR in mice after PHx. Results from RNA expression data from clinical samples and proteomic analysis from liver tissues indicated that peroxisome proliferator-activated receptor α (PPARα)-mediated fatty acid metabolism pathway were crucially associated with the TSG-provided promotion on LR. TSG enhanced the nuclear translocation of PPARα and the mRNA expression of a series of PPARα-regulated downstream genes. In addition, TSG lowered hepatic triglyceride (TG) and non-esterified fatty acid (NEFA) amounts and increased hepatic adenosine triphosphate (ATP) level in mice after PHx. TSG up-regulated the transcriptional activity of PPARα in vitro. Next results displayed that TSG promoted cell proliferation as well as ATP level in mice primary hepatocytes, which were abolished when PPARα was suppressed. Meanwhile, the cell viability was also elevated in mice primary hepatocytes treated with ATP. ConclusionActivating PPARα-mediated fatty acid β-oxidation (FAO) pathway led to the production of ATP, which contributed to the TSG-provided promotion on LR after PHx in mice.

P-134 Fatty acid supplementation during warming improves pregnancy outcomes after frozen blastocyst transfers: a propensity score-matched study

Abstract Study question Does warming with fatty acids (FAs) affect the viability of human blastocysts and pregnancy outcomes after a single vitrified-warmed blastocyst transfer (SVBT)? Summary answer FA-supplemented warming solutions promoted the blastocyst outgrowth by decreasing the degeneration of trophoblast cells and improved pregnancy outcomes after SVBTs. What is known already Vitrification is considered a well-established technique and has been used worldwide. However, previous studies demonstrated the detrimental effects of vitrification on pre-implantation development and term pregnancy in oocytes and embryos. Vitrification reduces the intracellular lipid content and decreases the competence for pre-implantation development. The FA supplementation into the warming solution restores the lipid content in the ooplasm and improves developmental competence by promoting the β-oxidation pathway in mice. Although, in humans, the FA-supplemented warming solutions reportedly improved pregnancy outcomes after vitrified-warmed cleavage-stage embryo transfers, the effectiveness of these solutions on blastocyst warming remain unclear. Study design, size, duration The experimental study included 401 discarded blastocysts. The blastocysts were warmed using the FA-supplemented (FA group) or non-supplemented (control group) solutions. The blastocyst adhesion, outgrowth area, and incidence of outgrowth degeneration were assessed. The retrospective cohort study included 4,426 treatment cycles from 4,426 women who underwent SVBTs between January 2023 and August 2023 (January–May 2023, control group; June–August 2023, FA group). Pregnancy outcomes were compared statistically between the groups. Participants/materials, setting, methods The implantation competence was experimentally examined using an outgrowth model. The blastocysts were placed on fibronectin-coated dishes and cultured for 96 hours. The embryos were designated as degenerated when the outgrowth area decreased by &amp;gt; 10%, compared with the maximum area. For the clinical study, multiple covariates that affected the outcomes were used for propensity score matching. The patients in the FA group were matched with controls using a propensity score in a 1:1 ratio. Main results and the role of chance The adhesion rate after the outgrowth culture was comparable between the control and FA groups. However, the outgrowth was larger in the FA group than in the control group. The multivariate linear regression analysis also demonstrated similar results. Furthermore, the incidence of outgrowth degeneration was significantly decreased in the FA group. Furthermore, the subgroup analyses revealed that FA-supplemented warming solutions were effective in blastocysts donated from young patients, day 5 blastocysts, and morphologically good blastocysts. For the retrospective cohort study, the implantation, clinical pregnancy, and ongoing pregnancy rates were significantly higher in the FA group than in the control group. The multivariate logistic regression analysis also demonstrated that warming with FA-supplemented solutions significantly increased the probability of ongoing pregnancies. However, the rates of early pregnancy loss and miscarriage in the first trimester were comparable between the groups. The subgroup analyses demonstrated a similar trend to in vitro experiments; the FA-supplemented solutions were more effective in good prognosis cases (young patients, day 4/5 blastocysts, and morphologically good blastocysts) than in poor prognosis cases (advanced age patients, day 6/7 blastocysts, and morphologically poor blastocysts). Limitations, reasons for caution This was a single-centre, retrospective study, necessitating further multicentre studies to ascertain the generalizability of these findings to other clinics with different protocols and/or patient demographics. Despite using propensity score matching and multivariate models to adjust for the confounders, we cannot adjust for all possible confounders. Wider implications of the findings FA-supplemented warming solutions improved pregnancy outcomes after SVBTs, particularly in patients with a good prognosis. These warming solutions could contribute to maximizing clinical outcomes, shortening treatment periods, and reducing patient burden. Trial registration number not applicable